Alcohol abuse is a significant contributor to increased morbidity in orthopedic trauma cases, leading to increased cases of fracture malunion and nonunion. Others have shown that alcohol use is associated with poor bone health, and our laboratory has shown that acute and chronic EtOH administration significantly impairs fracture healing in rodent models. However, the exact mechanism behind these effects of EtOH remains unknown. Previous studies conducted in our laboratory have shown that the canonical Wnt signaling pathway is perturbed by EtOH administration. This pathway has now become of great interest to us because it is a key pathway in driving mesenchymal stem cell (MSC) differentiation to osteoblasts and chondrocytes, two key cell types in maintaining normal bone homeostasis and highly important in the fracture healing process. It is well established that the metabolism of EtOH can increases cellular and systemic oxidative stress, and we have shown that the administration of a potent anti-oxidant can reverse the disruptive effects EtOH exhibits on fracture healing. In addition to this, there is a family of transcription factors known as the FoxO transcription factors that are known to be activated by oxidative stress, and once activated they bind to free -Catenin, the end effector molecule of the canonical Wnt pathway, which FoxO transcription factors need as a cofactor to bind to and upregulate specific target genes. Taken together, FoxOs appear to be a mechanistic link between the effects mediated by alcohol-induced oxidative stress and the perturbation of Wnt signaling. It is our hypothesis that alcohol exposure creates an environment of elevated oxidative stress inside MSCs at the fracture site and callus, leading to activation and upregulation of FoxO transcription factors that will perturb Wnt signaling by competing for -catenin, thus leading to improper differentiation of MSCs and poor fracture repair. To test this hypothesis we have established two aims. Aim 1 will determine if alcohol-induced oxidative stress is antagonizing normal Wnt signaling and normal fracture healing through activation of FoxOs in the fracture callus. Aim 2 will elucidate if FoxO-mediated oxidative stress signaling is having an effect on MSC differentiation in vitro. Studies will be carried out using both an in vivo mouse model of alcohol administration, and in vitro cultured MSCs. We have already been able to show that FoxO3a, vital in upregulating genes to combat rising oxidative stress, is elevated in the fracture callus of mice two days after EtOH administration and fracture by qRT-PCR. The FoxO3a target gene Catalase was also upregulated. These results were recapitulated in cultured MSCs exposed to EtOH, in which we measured increases in FoxO3a expression as well as Catalase. Overall, this study stands to help elucidate the problematic mechanism behind alcohol- disrupted fracture healing, which can benefit the knowledge of basic bone and fracture healing biology, and ultimately have magnanimous benefits overall to the treatment of orthopedic trauma and fractures. PUBLIC HEALTH RELEVANCE: Alcohol use is known to be detrimental to bone health and perturb fracture healing, yet it is unknown how alcohol exhibits these effects. This study proposes to discover the mechanism behind alcohol's actions on bone and fracture repair biology, eventually leading to preventative and therapeutic treatments of alcohol- induced osteopenia and fracture malunion.